Synergistic performance of triggered drug release and photothermal therapy of MCF7 cells based on laser activated PEGylated GO + DOX

: Graphene oxide is used as a singular 2D nano-carrier in cancer therapy. Here, graphene oxide is used as a hybrid chemo-drug graphene oxide (GO) + doxorubicin (DOX), mainly due to its unique chemical and optical properties. The laser triggers GO + DOX for selective drug delivery to optimize the drug release. The characterization of GO is investigated in terms of laser properties at 808 nm. Furthermore, the laser activates GO + DOX compounds to treat MCF7 cancerous cells. The drug release strongly depends on the temperature rise that mainly eﬀects on the viability of the cancerous cells of interest. DOX simultaneously acts as a chemo-drug and as an optical ﬂuorescent agent, whereas GO performs as an eﬃcient photothermal nano-carrier. In fact, the GO-DOX hybrid drug demonstrates multifunctional during malignant cell treatment. We have shown that the laser heating of GO enhances the release percentage up to a treatment yield of 90%. This arises from the synergistic nature of DOX and GO compounds in simultaneous chemo/photo thermal therapy. Furthermore, the ﬂuorescence property of DOX is used to assess the GO uptake using confocal microscope imaging.


Introduction
Cancer is one of the most lethal diseases affecting the world today. The uncontrolled division of abnormal cells in an organ is what results in the high global death rate [1,2]. In practice, the chemotherapy has been so far an effective method after surgical resection [3]. Inadequate dosage, incompatibility and resistance to chemo-drugs are among its drawbacks. This method also suffers from serious side effects on healthy cells [4]. Recently, serious attempts have been made to temper undesired chemotherapeutic cellular damages. One solution relies on drug loading of the appropriate nano-carriers based on selective drug delivery; protecting the healthy tissue from unwanted damage [5]. Nano-carriers such as liposomes, polymers and carbon nanoparticles are alternatives to target the cancerous cells due to careful control of drug release [6,7]. The proper selection of efficient carbon based nanoparticles is a challenging issue. Carbon nanostructures as potential nano-carriers are known to be beneficial in drug delivery. Not only the nanoparticles are expected to carry optimal quantities of the drug molecule, but will also be able to localize release of optimal dosage into abnormal cell regions. Among these carbon nanostructures, GO exhibits an outstanding potential for drug delivery due to its unique characteristics. The properties include high effective area, exceptional electronic conductivity, good thermal conductivity, strong mechanical strength, inherent biocompatibility, scalable production, facile biological/chemical functionalization, and low cost [8][9][10]. The properties relating to biomedical applications of graphene have been investigated extensively, including its capacity for drug/gene delivery, biological sensing and imaging, its antibacterial application, as well prospects as a biocompatible scaffold for cell culture growth [11]. Nanoscale graphene oxide (NGO) has been shown to be an efficient nano-carrier for the drug delivery of water insoluble, aromatic, anticancer drugs, directly into cells [12]. So, NGO was conjugated with an amine-terminated six-armed polyethylene glycol (PEG) molecule. On the other hand, SN38 is an antineoplastic drug which is the active metabolite of irinotecan but has 1000 times more activity than irinotecan itself. after SN38 loading onto the NGO surface by means of simple non-covalent adsorption via π-π stacking and then the functionalized nanoparticle, exhibits high cytotoxicity against HCT-116 cancer cells. Moreover, the delivery of chemotherapy drugs can be targeted directly into cancerous cells by a Rituxan (CD20+antibody) conjugated NGO-PEG [13]. pH-sensitive drug release has also been studied for GO-based systems [14][15][16]. Subsequently, thermo-responsive drug delivery based 2D materials has been examined [17]. GO can be photo-activated for photothermal treatment thanks to its strong optical absorbance, particularly across the near-infrared (NIR) spectral range within the therapeutic window (700-1100 nm) [18]. Furthermore, in vivo tumor uptake at 808 nm has been examined for its potential in GO-PEG photo therapy [19]. The evidence of highly efficient tumor damage has been observed under low power exposure treatment with no side effects occurring in the test-mice. This attests to the desirable biocompatibility accompanying the beneficial photothermal response [20]. Moreover, the synergistic effect during simultaneous chemo-photothermal therapy has been realized by exiting GO through exposure to diode laser radiation at 808 nm [21]. The pulsed laser at 808 nm was utilized to trigger typical Rd6G release into carcinoma tissues [22] manipulated by varying multiple laser parameters including fluency, spot size, and shot number. In the case of laser exposure below threshold (16 J/cm 2 ), corresponding to temperature < 4°C, the release process became ineffective indicating an insufficient energy content to break R6G-rGO linkages.
In this work, DOX loaded on GO-PEG is examined as a hybrid chemo drug, activated by light from a continuous wave (CW) GaAs laser at 808 nm. GO heating by laser is a dominant mechanism that gives rise to elevated local temperatures, up to 45°C where the drug delivery is initiated. For the purpose of initiating hyperthermia, the local temperature should reach between 35-80°C. The individual and synergistic effects of chemo and hyperthermia treatments are measured on MCF7 cancerous cells and their viability is verified. In fact, a series of experiments have been carried out on DOX, GO, and GO + DOX without light activation (pristine) and with laser excitation.
A Jenway 6715 spectrophotometer was employed to record UV-Vis spectra ranging 190-900 nm with 0.4 nm resolution. A 2 W-CW GaAs laser OEMDL-808(FC) by OElabs, was selected with good stability to carry out the laser exposure on the samples. A confocal microscope (Nikon) and an inverted microscope (Optika) were exploited for high resolution fluorescence imaging.
The release percentages are calculated using UV-Vis spectrometer according to the material incubation at two different pH conditions (5.5 and 7.5) for different time intervals (30 min-48h) at 37°C. At first, we remove each sample at certain time, then centrifuging the suspension to measure the UV-Vis absorbance of DOX (supernatant) to give out rerelease percentage via the calibration graph.

Preparation of PEGylated GO (GO-PEG)
GO-PEG was prepared similarly to Ref. [23]. Figure 1 illustrates the mechanism of GO PEGylation. 72 mg of NaOH is added into 2 ml of GO (2 mg/ml) and sonicated for 4 hours. Then, 0.4 ml of HCl is added to the solution and rinsed several times to remove the salts, giving the GO-COOH compound of interest. Afterwards, 25 mg PEG is added to the GO-COOH solution (1 mg/ml) and sonicated for 15 min, then 5 mg EDC (dimethylaminopropyl) carbodiimide-ethyl-3-(3-1) is injected into the solution. The latter is homogenized for 30 min using the bath sonicator. In the next step, another 5 mg of EDC is recharged to the solution and sonicated for 20 min. Then, it is stirred overnight at room temperature. Subsequently, any possible aggregates are removed by centrifuging at 21,000g for 30 min. Then, the supernatant is collected and dialyzed against any excess PEG and EDC to be ready for further treatment.

DOX loading on the GO-PEG
DOX is easily loaded onto GO-PEG by mixing DOX with GO-PEG at pH 8.5 stirring overnight [24]. The unbounded DOX is removed by centrifuging at 21000 g for 10 min. The yield of DOX entrapment is nearly equivalent to ∼ 90%. The GO spectrum is characterized by a single peak at 230 nm corresponding to the π→π* molecular transition which shifts to longer wavelengths in the case of GO-COOH. The GO-PEG absorbance is higher than GO over the UV-Vis spectrum because of the recovery of π stacking [20] and its brown color appears to darken as shown in Fig. 1. Hence, not only does the PEGylation improve the GO biocompatibility [11], but also significantly enhances the spectral absorbance. Figure 2(b) depicts the Zeta potential of the two compounds, indicating the GO Zeta potential is much higher than that of GO-PEG [25].  with GO functional groups. In addition, the bands at 3507 cm −1 is revealed due to N-H stretched GO-PEG vibrations [12]. Figure 2(d) presents the size distribution of GO. The mean lateral size were measured ∼ 37 µm.

DOX release
The drug release is dependent on the pH of the suspension in the loaded DOX on GO-PEG compound. Figure 3 illustrates the instantaneous release in 48 hrs follow-up at pH 5.5 and 7.5. The DOX release is almost 5% at pH=7.5 (similar to blood condition), whereas it rises up to 28% at pH=5.5 (acidic environment in breast cancer) hence DOX experiences higher rates of release from GO-PEG under acidic conditions [26]. This emphasizes that the pH dependence is low during the release process of the loaded DOX, however it is essential to employ an external agent to provide a stimulus and enhance the release rate. The temperature rise can elevate the release rate, because the π-π stacking bonding are mostly broken at high temperatures. GO-PEG displays a favorable spectral absorbance at 808 nm according to Fig. 2(a). Here the higher release rate of drug molecules takes place by GO-PEG nano-carriers photo stimulated, using GaAs diode laser. In fact, the combination of photo-chemo therapies together provides a notable synergistic effect to enhance the yield of treatment.

Laser exposure on GO-PEG
The temperature rise of the GO-PEG depends on its concentration and laser properties such as average power and exposure time. Figure 4 depicts the temperature rise of GO-PEG in terms of the exposure time at various laser powers. In general, GO-PEG does show a certain temperature rise in terms of concentration, laser power, and irradiation time. In this regard, the π-π stacking bonds are broken at temperatures higher than 40°C [22]. Therefore, the toxicity scales up for concentrations larger than 0.1 mg/ml and laser power above 150 mW (equivalent to 19 W/cm 2 power density).   Figure 5(b) depicts the cell temperature rise as a function of laser power at 0.73, 1.5 and 2 W for the corresponding irradiation of 3, 6 and 10 min. In fact, no notable toxicity takes place for the treated cells under low power exposure with viability higher than 90%. Figure 5(c) plots the viability of cancerous and normal cells in terms of laser power emphasizing slight cell mortality especially at low laser power. It is worth noting that the damage threshold of normal cells is lucidly higher than the malignant ones according to Ref. [27]. Normal cells survive at higher temperatures than cancerous cells without irreversible damage. This is a key feature of the method to kill cancerous cells while protecting the healthy cells. Hence, the laser with given properties at 808 nm does induce negligible damage on the normal cells, while lethal events take place against cancerous cells. Furthermore, Figs. 6(a) and 6(b) displays that toxicity of DOX and the corresponding value IC50 = 0.0016 mg/ml.  Figures 7(a) and 7(b) illustrates the release percentage and temperature rise versus laser power for 0.3 and 0.5 mg/ml of GO-PEG-DOX. The compound usually breaks at higher temperatures mainly due to π-π stacking [22]. At temperatures above 50°C, the release rate starts reducing (Fig. 7(c)) due to dissociation of π-π stacking among the GO-PEG layers leading to the reduction of the layer spacing [24]. This may increase the aggregation of GO-PEG, leading to the enhancement of DOX trapping between layers.

Laser triggering and drug release
The temperature rise looks like to aggregate GO-PEG layers due to the removal of functional groups such as hydroxyl [20]. In this case, the DOX molecules are supposed to be trapped between the layers of GO nanostructures resulting in the slow-down of the drug release rate.

Synergistic effects in simultaneous chemo/photothermal therapies
To summarize our findings in a single frame, Fig. 8 illustrates the viability for various combinations of GO-PEG-DOX under laser exposure at various concentrations. At first, the control sample is examined which does contain no additive conjugation with 100% viability of cancerous cells. Simulating the traditional cancer therapy, DOX chemo-drug is injected to the cells for 24 h with 50% viability. Later, DOX chemo drug is loaded on GO at two concentrations; 0.3 and 0.5 mg/ml without laser exposure.
The cells undergo a notable reduction of viability of ∼ 37%. Subsequently, the cancerous cells are examined with GO under certain laser exposure (typically 6 min, 0.73 W and 3 min, 1.3 W).
The same temperature rise up to 50°C gives rise to drastic decrease in viability (18%) emphasizing the importance of the photothermal effect in drug delivery. This demonstrates that the cell mortality due to the photothermal effect is notably higher than that of DOX or GO + DOX without exposure as predicted. Finally, the effect of hybrid GO + DOX on MCF7 cells is investigated under certain laser exposure conditions; to demonstrate a drastic fall in viability below 10% and achieve a successful cancer treatment as the invert microscopic images confirm them to the Figs. 8(b)-8(e). This result is due to the simultaneous synergistic effects of chemo and photothermal therapies. We believe that the photothermal therapy is dominant at high laser exposures, while the chemotherapy is a desirable mechanism at low flounces. Here, DOX acts as both fluorescent chemo drug and toxic chemical agent at the same time [28,29]. The confocal microscopic imaging based on DOX fluorescence properties is used to verify the status of cell mortality. As a consequence, GO-PEG acts as a good quencher [30,31] and nano-carrier to deactivate the excited fluorophores as long as DOX is loaded on GO-PEG. During delivery, there is a condition (temperature > 50°C) where the detachment of DOX from GO takes place. The DOX release allows the chemo-drug to act as an efficient fluorophore for fluorescence imaging and monitoring of the cells. Figure 9(a) illustrates the absorption and emission spectra of DOX. The Nd:YAG laser is exploited at 532 nm for provoking DOX and the diode laser is used at 405 nm for excitation of DAPI. An interference filter is situated to transmit the DOX fluorescence emission ranging 560-610 nm and similarly for DAPI fluorescence ranging 425-475 nm. The laser emission are blocked too. Figure 9(b) represents the confocal image with high background because the DOX release resembles to be low after 4 h in agreement to Fig. 3. In fact, the fluorescence emission of loaded DOX is quenched by GO-PEG conjugate. Figures 9(c) and 9(d) illustrate the uptake of GO-PEG + DOX after 24 h. Therefore, due to the elevation of DOX release, the fluorescence intensity intracellularly is increased with low background. Furthermore, it can be observed that after 24 h incubation with DOX, the cell cytoplasm displays orange fluorescence, indicating DOX enters into live cell and located in the cell cytoplasm. Hence, DOX as a fluorescent chemo drug [29] can be used for the purpose of real time cancer therapy and fluorescence imaging at the same time.   [29]. Confocal microscopic image to verify MCF7 cell after (b) 4 h and (c and d) 24 h. The drug release level is low after 4 h such that GO-PEG quenches the fluorescence emissions of loaded DOX. Note that (b) shows an image with high background. The uptake of (GO-PEG + DOX) after 24 h elevates the release of DOX intracellularly. Therefore, (c) and (d) change to low background. The blue color in (c) addresses the cell nucleus that was pigmented by DAPI and the orange color in (c) and (d) indicate the fluorescence emission of DOX during the release inside the cells.

Conclusion
This is a continuation of our previous works on the fluorescence properties of a variety of chemo drugs in order to achieve the optimal conditions i.e. exposure time and laser power during chemo drug release in the selective area. The application of lasers at wavelengths near the absorption peak of GO-PEG increases the yield of heating. However, we intend to apply the heating method for in-vivo human treatment. So, it is very important to use laser beam that benefits more penetration depth with minimal damage for healthy tissues in surroundings. Near infrared range (800-1100 nm) acts as therapeutic window to be very useful for this purpose because those show low absorption and more penetration depth in tissues. As a consequence, a diode laser at 808 nm was employed to irradiate the samples with adequate photo thermal yield.
Drug release in the infected organ takes place as a result of adequate temperature rise and the acidity of the tumor environment. DOX is loaded on the GO-PEG nano-carrier and irradiated by a diode laser at 808 nm to activate the extra cellular drug release to targeted cancerous cells. The release rate can be controlled by tuning the laser parameters. Then the DOX molecules inside the cells are activated through heat generation. Furthermore, laser exposure allows a combination of specific drug delivery and the photo-thermal effect on the target. In fact, the release rate can be controlled by laser parameters, GO population, its absorbance and the laser properties. By making use of GO photo excitation, the release rate and efficiency can be enhanced leading to effective cell mortality. It is shown that the temperature, nonlinearly increases in terms of the laser power and the optimal release rate is achieved in the 42-52°C interval.
In a series of experiments, the MCF7 cell viability is assessed for control (100%), DOX chemodrug (50%), GO + PEG + DOX (37%), laser activated GO + PEG (18%) and laser activated GO + PEG + DOX (<10%). We have shown that the photothermal therapy is dominant at high laser exposures, while the chemotherapy would be a desirable mechanism at low flounces. It is concluded that the synergistic effect of chemotherapy and photo thermal therapy is realized when GO, PEG, and DOX work in conjunction (GO + PEG + DOX) resulting in the majority of cancerous cell population being killed (> 90% mortality). Confocal microscopy clearly verifies the DOX penetration into cells using its fluorescence property; attesting to the use of the hybrid drug of interest in favor of simultaneous diagnosis and treatment.
According to the successful preliminary result reporting here, we plan carrying out in vivo study of laser activated chemo-photo thermal therapy of cancerous breast tissues through the enhanced permeability and retention (EPR) effect. Then, it is anticipated to reduce the chemical side effects, emphasizing more contribution of GO 2D material in the localized thermal therapy. Hence, this minimally invasive therapy will be clinically developed in practice.